GSM6620670: Guide targeting Gmnc, mTECs at air/liquid interface for 5 days, replicate 1; Mus musculus; RNA-Seq
On day five at air/liquid interface, mTECs were washed with PBS three times both apically and basally. Trypsin-EDTA at a concentration of 0.05% in Cell Dissociation Buffer (Gibco, Cat #13151014) was added both apically and basally and cells were incubated at 37°C for 10 min. Cells were lifted from the membrane with gentle pipetting and trypsin deactivated in mTEC Basic with 2% Nu-Serum (Corning, Cat #355100). Fresh 0.05% Trypsin-EDTA in Cell Dissociation Buffer was added to the apical surface and incubated at 37°C for 5 min. The pipetting, inactivation, and 5 min incubation steps were repeated twice. mTECs were filtered through a 100 uM cell strainer, washed and resuspended in 0.04% BSA in PBS at 1000 cells per uL to prepare for scRNA-seq. Library was performed according to the manufacter's instructions (Chromium Single Cell 3' Next GEM Reagent kit protocol, 10x Genomics). Briefly, mTECs were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.